Generation of OVA-specific IgG antibodies via mouse

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Generation of OVA-specific IgG antibodies via mouse

Introduction

Immunization, and immunological analysis of specific antibodies to specific antigens is a vital component of both research and biomedical applications. Mice are widely available laboratory animals that can easily be manipulated and used for the production of Abs against a broad range of Ags, using well-defined immunization protocols. Such techniques, using laboratory animals, allow optimal in-vivo affinity maturation of the Abs, subsequent to multiple immunizations, followed by analysis. (1)

Aluminum-containing adjuvants have been historically serving both research and analytical process as immunopotentiators in vaccines and continue to be the most widely used clinical adjuvants. Classically, it has been shown in vitro that in the absence of the alum, the Ags were simply taken up by the lymph node (ND) DC’s, whereas subsequent to the adjuvant stimulation, the Ag was taken up and processed and presented on the surface of the monocytes for recognition. Despite the fact that the aluminum-containing adjuvants have been used in both experiments and clinical research, it is unexplainable as to the reason why there is no consensus regarding the full mechanism of action by which it induces an immunological response. (1)

The alum adjuvant is combined with Ovalbumin (OVA), which gives it its popular name OVA-alum Ag. OVA is the major protein constituent of chicken egg whites, thus being a derived glycoprotein that is adequately large and complex to be mildly immunogenic. Accordingly, OVA is widely used as an Ag for laboratory research experiments. (2)

The invention of the vaccinations was at the highlight of the war between microorganisms and humans. The Ag OVA-Alum is a widely used Ag that proliferates an immunological response within mice, thus can be categorized as an immunization. Prior research has identified a crucial role for the innate immune system in recognizing vaccines and adjuvants in order to induce appropriate protective immune responses. The innate system can further sense microorganisms through pattern-recognition receptors (PRRs), such as the Toll-like receptors (TLRs), which are being expressed most, including dendritic cells (DCs). Once the DC is in the spleen, it’s looking for an Ag to bind to, thus can secrete cytokines that will signal the follicular helper T cells (TFHcells) to become active. Classically, TFHcells produce interleukin 21 (IL-21) and which drives the differentiation of B cells within the germinal centers and generate memory B cells. In addition, differentiating memory CD4+and CD8+T cells can be subcategorized into central memory and effector memory cell subsets, each with a distinct functionality.(2)

Subsequent to creating an immune response, a method to analyze the Abs is required. ELISA is a widely used tool in both in diagnostic studies, and in research as an analytical tool for the detection and quantification of specific Ags or Abs in a sample. ELISA uses the basic immunological notions of an Ag that binds to its specific Abs within the sample.(3) This test is based on the interaction of specific Abs with plastic-bound (insolubilized) Ag. This is followed by an amplification of the reaction with an enzyme-conjugated second Ab, which is directed against the Fc portion of the first Ab. (4) A chromogenic substrate for the enzyme yields a color change to indicate a presence of an Ag. Qualitative or further quantitative measures can be evaluated based on such colorimetric reading.(3)

In this study, we demonstrated that proliferation of memory B cells that occurs when the mice are immunized with OVA-Alum Ag, on two different occasions, the second being the booster shot. Subsequent to the proliferation, the aim was to develop and analyze qualitatively and quantitatively the binding of the OVA-specific IgG to the Ags that were insoluble in the ELISA plate using colorimetric readings.

Methods

The procedures used were adapted from the MI-3600G Lab Manual, page 6-19. (4) All experiments performed with the mouse have ethics approval by Western University.

There was only once change within our protocol, being a change in the dilutions from 1:50 to 1:60.

Results

The primary immunization of the BALB/c mouse with OVA-Alum antigen produced a higher immunological IgG response to the antigen compared to the booster shot immunization with the OVA-Alum antigen

The purpose of this experiment was to measure and quantify the whether or not two consecutive immunizations yield a larger amount of OVA-specific IgG’s compared to a saline booster. The readings of the OD405 at a declining concentration that is known of the anti-OVA IgG Ab in ELISA plate wells (n=3). The value of each mean OD450 triplicate was corrected and plotted in order to generate a standard curve and a linear regression relationship. The standard curve can be used as a reference in order to determine the concentration that is unknown of the OVA-specific IgG in the BALB/c mouse plasma sample. (Fig.1A) A linear regression of the standard curve for figure 1B concentrations of OVA-specific Abs was created via a logarithmic scale in order to generate a linear equation to obtain the unknown concentration of BALB/c mice OVA-specific IgG antibodies. The OVA-specific IgG was serially, from an initial concentration of 400 ng/mL to a final concentration of 1.5625 ng/mL from 8 sequential rows in three columns on the ELISA plate, each with a coating of 10 µg of OVA.

""A.

B.

""

Figure 1. The OD readings are directly proportional to the concentration of the OVA-specific IgG. A) OVA-specific IgG was diluted serially, from an initial concentration of 400 ng/mL to a final concentration of 1.0675 ng/mL from 8 sequential rows in three columns on the ELISA plate, each with a coating of 10 µg of OVA. The resulted standard curve for each of the diluted samples indicates the concentration of the IgG within the sample, as well as the OD405 that is correspondent to it. B) The equation produced via a logarithmic scale was a linear regression, with no outliers being discounted. For both, Figure A and B, results obtained were mean±SD of the concentrations of the Abs.

Table 1. – The results of the immunizations procedures using the BALB/c mouse with corresponding ELISA plate results.

BALB/c Mouse Immunization Protocol

BALB/c Mouse serum Dilutions

Corrected- OD405 of ELISA wash buffer

IgG concentration in diluted Mouse Plasma

IgG Concentration in Neat plasma

OVA+OVA

1st dilution

25µL serum to 475µL isotonic saline

DF: 20

0.121

DF 200: .128µg/mL

25.6µg/mL

2nd dilution

DF: 5

Final Dilution

100

OVA+ saline

DF

60

DF 240: .159 µg/mL

38.2 µg/mL

""Figure 2. The concentrations of OVA primary + booster IgG are evidently higher in the neat mouse plasma than the concentrations of the OVA + saline. 1) The concentrations, in µg/mL, of OVA+OVA were measured from a dilution factor of 200, form the 3 samples. 2) The concentrations of OVA + Saline were measured using a dilution factor of 240 form the 3 samples of the neat mouse plasma.

1.Kool, M., T. Soullie, M. van Nimwegen, M. A. Willart, F. Muskens, S. Jung, H. C. Hoogsteden, H. Hammad, and B. N. Lambrecht. 2008. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. The Journal of experimental medicine 205: 869-882.

2.Pulendran, B., and R. Ahmed. 2011. Immunological mechanisms of vaccination. Nature immunology 12: 509-517.

3.Gan, S. D., and K. R. Patel. 2013. Enzyme immunoassay and enzyme-linked immunosorbent assay. The Journal of investigative dermatology 133: e12.

4.Summers, K. 2013-2014. Laboratory Techniques in Microbiology and Immunology . Immunology Laboratory Manual. Western University, London, CA.

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